Ribonucleotide reductase inhibitors and methods of use

ABSTRACT

Provided herein are novel compounds that inhibit ribonucleotide reductase (RR) by binding to RRM2 and interfering with the activity of the RRM1/RRM2 holoenzyme. These inhibitors may be used to inhibit RR activity and to treat various conditions associated with RRM2 expression, such as for example certain cancer types, mitochondrial diseases, or degenerative diseases.

RELATED APPLICATIONS

The present utility application is a divisional application of U.S.patent application Ser. No. 12/420,713, filed Apr. 8, 2009 now U.S. Pat.No. 7,956,076, which claims priority to U.S. Provisional PatentApplication No. 61/043,372, filed Apr. 8, 2008, the disclosures of whichare incorporated by reference herein in their entirety.

GOVERNMENT INTEREST

This invention was made with Government support under grant numberCA127541 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND

Ribonucleotide diphosphate reductase (RR) is a highly regulated enzymein the deoxyribonucleotide synthesis pathway that is ubiquitouslypresent in human, bacteria, yeast, and other organisms (Jordan 1998). RRis responsible for the de novo conversion of ribonucleotide diphosphateto 2′-deoxyribonucleotide diphosphate, a process that is essential forDNA synthesis and repair (Thelander 1986; Jordan 1998; Liu 2006). RR isdirectly involved in tumor growth, metastasis, and drug resistance (Yen1994; Zhou 1995; Nocentini 1996; Fan 1998; Zhou 1998).

The proliferation of metastatic cancer cells requires excess dNTPs forDNA synthesis. Therefore, an increase in RR activity is necessary as ithelps provide extra dNTPs for DNA replication in primary and metastaticcancer cells. Because of this critical role in DNA synthesis, RRrepresents an important target for cancer therapy. However, there hasbeen little progress in the development of RR inhibitors for use incancer treatment. The three RR inhibitors currently in clinical use(hydroxyurea, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, andGTI2040) each have significant drawbacks. Therefore, there is a need inthe art for more effective compositions and methods for targeting andtreating RR-based cancers.

SUMMARY

In certain embodiments, a novel set of compounds including COH4, COH20,and COH29, as well as various chemical derivatives thereof, areprovided. Also provided are compositions and pharmaceutical formulationscomprising these compounds.

In certain embodiments, methods are provided for inhibiting RR activityin a cell by contacting the cell with one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

In certain embodiments, methods are provided for inhibiting the growthor proliferation of a cell expressing RRM2 by contacting the cell with atherapeutically effective amount of one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

In certain embodiments, methods are provided for treating cancer in asubject in need thereof by administering a therapeutically effectiveamount of one or more of the compounds provided herein, including COH4,COH20, and/or COH29. In certain embodiments, the cancer may becharacterized by RRM2 overexpression, and in certain embodiments thecancer may be resistant to treatment with hydroxyurea.

In certain embodiments, methods are provided for inhibitingproliferation of a stem cell expressing RRM2 by contacting the stem cellwith a therapeutically effective amount of one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Suppression of RRM2 via siRNA in mice implanted with human HepG2liver cancer cells decreases tumor growth.

FIG. 2: RRM2 overexpression in cancer tissues and cell lines incomparison to normal tissue.

FIG. 3: RRM2 transfectant enhances invasive potential in human cancercell lines.

FIG. 4: Prediction of V-shaped ligand binding pocket on RRM2. Ironclusters are shown in red.

FIG. 5: Synthesis strategy for NCI-3 analogs

FIG. 6: Inhibition of RR activity in vitro by HU, 3-AP, NCI-3, COH4, andCOH20.

FIG. 7: Inhibition of intracellular RR activity by COH20.

FIG. 8: Inhibition of dNTP pools by COH20 in KB cells.

FIG. 9: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human prostateLNCaP cancer cells in vitro.

FIG. 10: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human KB cancercells in vitro.

FIG. 11: Cytotoxicity of 3-AP, HU, COH4, and COH20 in normal humanfibroblast (NHDF) cells in vitro.

FIG. 12: Cytotoxicity of 3-AP, HU, COH4, and COH2 in human KBHUR cancercells in vitro.

FIG. 13: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human KBMDR(multidrug resistant) cells in vitro.

FIG. 14: Inhibition of human KB and KBMDR cell proliferation by COH20.

FIG. 15: Upper panel: Flow cytometry of KB cells following treatmentwith 3-AP or COH20. Lower panel: Annexin staining of KB cells followingtreatment with 3-AP or COH20.

FIG. 16: Single-dose pharmacokinetics of COH20 in rats.

FIG. 17: COH20 maximal tolerated dose determination in normal mice.

FIG. 18: Biacore analysis of RRM2 binding to COH20 and 3-AP.

FIG. 19: Interference of RRM1 binding to RRM2 by COH20.

FIG. 20: Inhibition of cancer cell growth by single-dose administrationof COH29.

FIG. 21: Inhibition of cancer cell growth by multiple-doseadministration of COH29.

FIG. 22: Inhibition of cancer cell growth by multiple-doseadministration of COH29. Results are grouped by cell line tissue oforigin.

FIG. 23: Inhibition of cancer cell growth by multiple-doseadministration of COH29.

FIG. 24: Inhibition of cancer cell growth by multiple doseadministration of COH29.

FIG. 25: Pharmcokinetic of COH29 in rats.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

The following abbreviations are used herein: 3-AP,3-aminopyridine-2-carboxaldehyde thiosemicarbazone; dNTP;deoxyribonucleotide triphosphate; HU, hydroxyurea; RR, ribonucleotidereductase.

The phrase “therapeutically effective amount” as used herein refers toan amount of a compound that produces a desired therapeutic effect. Theprecise therapeutically effective amount is an amount of the compositionthat will yield the most effective results in terms of efficacy in agiven subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20^(th) edition, Williams & Wilkins PA, USA) (2000).

“Treating” or “treatment” of a condition as used herein may refer topreventing or alleviating a condition, slowing the onset or rate ofdevelopment of a condition, reducing the risk of developing a condition,preventing or delaying the development of symptoms associated with acondition, reducing or ending symptoms associated with a condition,generating a complete or partial regression of a condition, curing acondition, or some combination thereof. With regard to cancer,“treating” or “treatment” may refer to inhibiting or slowing neoplasticand/or malignant cell growth, proliferation, and/or metastasis,preventing or delaying the development of neoplastic and/or malignantcell growth, proliferation, and/or metastasis, or some combinationthereof. With regard to a tumor, “treating” or “treatment” may refer toeradicating all or part of a tumor, inhibiting or slowing tumor growthand metastasis, preventing or delaying the development of a tumor, orsome combination thereof.

A cancer “characterized by overexpression of RRM2” as used herein refersto any cancer type that expresses RRM2 at either the mRNA or proteinlevel at a level greater than that of a corresponding normal cell ortissue. For example, a prostate cancer cell line is a cancercharacterized by overexpression of RRM2 if it expresses RRM2 at eitherthe mRNA or protein level at a level greater than that observed in acorresponding normal prostate cell. A cancer characterized byoverexpression of RRM2 as used herein also refers to any cancer type inwhich RRM2 inhibitors exhibit additional or selective effects comparedto normal, untransformed cells or tissues. For example, a cancer type isa cancer characterized by RRM2 overexpression if it has a greaterdependency on the nucleotide pool because of a difference in mitoticindices with normal cells, making it more sensitive to RRM2 inhibition.

dNTP production in eukaryotes is tightly regulated by RR, whichcatalyzes the rate-limiting step in deoxyribonucleotide synthesis(Jordan 1998). RR consists of a large subunit and a small subunit. Inhumans, one large subunit (RRM1, also referred to as M1) and two smallsubunits (RRM2, also referred to as M2, and p53R2) have been identified(Tanaka 2000; Liu 2006). The small RR subunits form two equivalentdinuclear iron centers that stabilize the tyrosyl free radical requiredfor initiation of electron transformation during catalysis (Ochiai 1990;Cooperman 2003; Liu 2006).

RRM1 is a 170 Kd dimer containing substrate and allosteric effectorsites that control RR holoenzyme activity and substrate specificity(Cory 1983; Wright 1990; Cooperman 2003; Liu 2006).

RRM2 is an 88 Kd dimer containing a tyrosine free radical and a non-hemeiron for enzyme activity (Chang 1979). p53R2 contains a p53-binding sitein intron 1 and encodes a 351-amino-acid peptide with strikingsimilarity to RRM2 (Tanaka 2000). RRM2 and p53R2 have an 80% similarityin amino acid sequence (Tanaka 2000).

p53R2 has been identified as a transcriptional target of p53 (Nakano2000; Tanaka 2000; Yamaguchi 2001), while RRM2 is transcriptionallyregulated by cell cycle-associated factors such as NF-Y and E2F (Filatov1995; Currie 1998; Chabes 2004; Liu 2004). Therefore, expression ofp53R2, but not RRM2, is induced by ultraviolet (UV) light,gamma-irradiation, or Doxorubicin (Dox) treatment in a p53-dependentmanner (Lozano 2000; Tanaka 2000; Guittet 2001). In p53 mutant ordeleted cells, RRM2 can replace p53R2 in the DNA repair process forcells exposed to UV irradiation (Zhou 2003; Liu 2005). P53R2 has beenfound to have a stronger reducing capacity than RRM2, which may providea chaperone effect in stabilizing p21 (Xue 2007).

It has been observed that the RB tumor suppressor suppresses RR subunitsas a mechanism for regulating cell cycle progression (Elledge 1990). RBinactivation, often observed in tumors, leads to increased dNTP levelsand a concomitant resistance of tumor cells to drugs such as5-fluorouracil (5-Fu) and HU (Angus 2002). While overexpression of theRRM2 subunit promotes transformation and tumorigenic potential via itscooperation with several activated oncogenes (Fan 1998), overexpressionof the RRM1 subunit suppresses malignant potential in vivo (Fan 1997).Increased expression of RRM2 has been found to increase thedrug-resistant properties of cancer cells and to increase invasivepotential, whereas RRM2 suppression leads to the reversal of drugresistance and results in decrease proliferation of tumor cells (Zhou1995; Huang 1997; Zhou 1998; Goan 1999; Chen 2000; Nakano 2000; Kuschak2002). Normal cells express very low levels of RR in non-proliferativestatus, whereas most neoplastic cells overexpress RR, thereby supplyingdNTP pools for DNA synthesis and cell proliferation. Thus, specificinhibition of RRM2 is likely to provide antineoplastic benefits.

Although RR represents an important target for cancer therapy, there areonly three RR inhibitors in clinical use: hydroxyurea (HU),3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine®),and GTI2040. HU, which blocks DNA synthesis by reducing the tyrosyl freeradical, has been marketed as a cancer therapeutic for over 30 years andis the only RR inhibitor that is commercially available (Nocentini1996). However, resistance to HU treatment is a common problem (Lassmann1992; Nyholm 1993; Le 2002). 3-AP, a small molecule iron chelatorinactivates RR, has been found to cause hypoxia, respiratory distress,and methemoglobulin of red blood cells. In addition, 3-AP selectivelytargets p53R2 instead of RRM2. GTI2040, an antisense molecule, has thusfar been ineffective in human trials. Other issues relating to thesethree RR inhibitors are incomplete RR blocking, short half-life, andregeneration of RR. In addition, mutation of p53R2 results in hereditarymitochondria depletion syndrome, but not cancer, and p53R2 knockout micedemonstrate kidney tubule disorder but no obvious cancer growth (Kimura2003). These observations suggest that RRM2 is responsible for tumorproliferation and metastatic potential, whereas p53R2 induced by DNAdamage signals for DNA repair. Therefore, an ideal RR inhibitor for usein cancer therapy would have greater potency than HU, less ironchelating ability than 3-AP, and specific targeting of RRM2.

As disclosed herein, RRM2 has been validated as an anti-cancer target.Suppression of RRM2 via siRNA was found to decrease tumor growth in miceimplanted with human HepG2 liver cancer cells. Expression of RRM2 wasdetermined to be significantly higher in cancer cells than incorresponding normal cells. In addition, human KB and PC3 cellstransfected with RRM2 exhibited increased invasive potential versustheir non-transfected counterparts, suggesting that RRM2 enhances theinvasive potential of cancer cells.

A diverse compound library from NCI Developmental Therapeutics Program(DTP) was screened to identify compounds that inhibit RR. Three of thefour compounds identified in this screen that inhibited RRM1/RRM2activity by 80% of more shared a similar structural scaffold, NCI-3.NCI-3 has the structure:

Initial hits from the screening process were synthetically andrationally optimized to obtain the RR inhibitors COH1, COH2, COH4,COH20, and COH29, the structures of which are set forth below.

Each of these compounds exhibited the ability to inhibit RR to asignificant degree, and COH20 and COH29 both exhibited the ability toinhibit cancer cell growth across a wide range of cancer cell types.Accordingly, in certain embodiments the present application disclosesnovel RR inhibitors, compositions, formulations, and kits comprising oneor more of these inhibitors, and methods of using these inhibitors toinhibit RR, inhibit cell growth or proliferation, treat cancer, and/orinhibit stem cell proliferation.

COH20 consists of three basic structural units: a pharmacophore (forinhibition of RR activity), a binding group (for selectivity), and alinking group (to connect the pharmacophore and the binding group. Asdisclosed herein, COH20 exhibited low micromolar range inhibition ofboth recombinant and intracellular RR in vitro, and caused a decrease indNTP pools. Biochemical analysis revealed that COH20 targets RRM2. Uponbinding the RRM1/RRM2 complex, COH20 appears to reside at the V-shapedpocket at the interface between RRM1 and RRM2 and block the free radicaltransfer pathway though a novel catechol radical stabilizationmechanism. Considering the size and chemical composition of COH20 andthe distance to the dinuclear iron center, the bound ligand (in thispocket) does not appear to be susceptible to iron chelation as with 3-APor involved in the direct quenching of the initially formed tyrosyl freeradical as with HU. COH20 was found to inhibit growth of the humanleukemia cell lines REH and MOLT-4, the human prostate cancer cell lineLNCaP, and the human oropharyngeal cancer cell line KB in vitro at aconcentration of less than 10 μM, while exhibiting less cytotoxicitytowards normal fibroblast cells than HU. COH20 also exhibited greatercytotoxicity towards the HU-resistant cell line KBHURs than 3-AP,indicating that it is capable of overcoming HU drug resistance. COH20exhibited cytotoxicity towards KBMDR cells at lower concentration than3-AP or HU (80 μM versus 200 μM and >1000 μM, respectively), indicatingthat COH20 circumvents MDR more effectively than 3-AP. In addition,COH20 had very low toxicity when administered to mice, with no evidenceor iron chelation or methemoglobulin.

As disclosed herein, COH29 showed promising growth inhibitory effects ona wide range of human cancer cell lines, including:

human non-small cell lung cancer cell lines NCI-H23, NCI-H522,A549-ATCC, EKVX, NCI-H226, NCI-H332M, H460, H0P62, HOP92;

human colon cancer cell lines HT29, HCC-2998, HCT116, SW620, COLO205,HCT15, KM12;

breast cancer cell lines MCF7, MCF7ADRr, MDAMB231, HS578T, MDAMB435,MDN, BT549, T47D;

ovarian cancer cell lines OVCAR3, OVCAR4, OVCAR5, OVCAR8, IGROV1, SKOV3;

human leukemia cell lines CCRFCEM, K562, MOLT4, HL60, RPMI8266, SR;

renal cancer cell lines UO31, SN12C, A498, CAKI1, RXF393, 7860, ACHN,TK10;

melanoma cell lines LOXIMVI, MALME3M, SKMEL2, SKMEL5, SKMEL28, M14,UACC62, UACC257;

prostate cancer cell lines PC3, DU145; and

CNS cancer cell lines SNB19, SNB75, U251, SF268, SF295, SM539.

In certain embodiments, COH29 showed a less than about 10 μM GI50 forthe NCI 60 human cancer cell lines, except colon cancer cell line HT29,melanoma cancer cell line UACC-257, ovarian cancer cell lineNCI/ADR-RES, and renal cancer cell line CAKI-1. In addition,pharmacokinetic studies of COH29 showed a dose-dependent manner whenCOH29 was administered by i.v. bolus.

COH4, COH20 and COH29 represent unique RR inhibitors with highantitumoral activity that provide significant advantages over previouslydisclosed RR inhibitors. Specifically, COH20 offers a unique mechanismand target specificity that interferes with the radical transfer pathwayat the RRM1/RRM2 interface with greater potency than HU and ameliorationof the iron chelation-related side effects observed with 3-AP.Therefore, provided herein in certain embodiments are small molecule RRinhibitors and methods of using these inhibitors to inhibit RR and totreat cancer. The inhibitors disclosed herein are capable of overcomingHU resistance, a common obstacle to cancer therapy, and are also capableof overcoming multidrug resistance.

In certain embodiments, a novel RR inhibitor as disclosed herein isCOH1, COH2, COH4, COH20, or COH29, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or prodrug derivative thereof.

In certain embodiments, a novel RR inhibitor as disclosed herein is:

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrugderivative thereof, wherein:

R₁ and R₂ are independently selected from the group consisting of alkyland aryl groups;

R₃ is selected from the group consisting of alkyl groups; and

X is selected from the group consisting of Br, halogen, alkyl and arylgroups.

In certain embodiments, an RR inhibitor as disclosed herein is:

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrugderivative thereof, wherein

X is selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups;

R₁-R₂ are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; and

R₁-R₂ may combine together to form a ring wherein the ring is aryl ornon-aryl.

As used herein, unless specified otherwise, the term “alkyl” means abranched or unbranched, saturated or unsaturated, monovalent ormultivalent hydrocarbon group. Examples of alkyl include, but are notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, ethenyl,propenyl, butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, dodecenyl, ethynyl, propynyl, butynyl,isobutynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl,undecynyl, dodecynyl, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, t-butylene, pentylene, hexylene, heptylene,octylene, nonylene, decylene, undecylene and dodecylene. In certainembodiments, the hydrocarbon group contains 1 to 30 carbons. In certainembodiments, the hydrocarbon group contains 1 to 20 carbons

As used herein, unless specified otherwise, the term “aryl” means achemical structure comprising one or more aromatic rings. In certainembodiments, the ring atoms are all carbon. In certain embodiments, oneor more ring atoms are non-carbon, e.g. oxygen, nitrogen, or sulfur.Examples of aryl include, without limitation, phenyl, benzyl,naphthalenyl, anthracenyl, pyridyl, quinoyl, isoquinoyl, pyrazinyl,quinoxalinyl, acridinyl, pyrimidinyl, quinazolinyl, pyridazinyl,cinnolinyl, imidazolyl, benzimidazolyl, purinyl, indolyl, furanyl,benzofuranyl, isobenzofuranyl, pyrrolyl, indolyl, isoindolyl,thiophenyl, benzothiophenyl, pyrazolyl, indazolyl, oxazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, thiaxolyl and benzothiazolyl.

As used herein, the term “pharmaceutically acceptable salt” means thosesalts of compounds of the invention that are safe and effective forapplication in a subject and that possess the desired biologicalactivity. Pharmaceutically acceptable salts include salts of acidic orbasic groups present in compounds of the invention. Pharmaceuticallyacceptable acid addition salts include, but are not limited to,hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,11-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe invention can form pharmaceutically acceptable salts with variousamino acids. Suitable base salts include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, anddiethanolamine salts. For a review on pharmaceutically acceptable saltssee BERGE ET AL., 66 J. PHARM. SCI. 1-19 (1977), incorporated herein byreference.

As used herein, the term “halogen” means F, Cl, Br, I and At.

The RR inhibitors disclosed herein specifically target RRM2, inhibitingthe interaction between RRM1 and RRM2 and inhibiting the activity of theRR complex. Therefore, these inhibitors may be used to certain cancers,including cancers associated with the overexpression of RRM2 or forwhich there exists a suitable therapeutic index. As disclosed herein,RRM2 may be overexpressed in breast cancer cells versus normal cells,and expression of exogenous RRM2 increases cancer cell invasivepotential. The inhibitors disclosed herein have been shown to inhibitgrowth of multiple cancer cell types in vitro, supporting the use ofthese inhibitors to treat a wide range of cancers.

Previous studies have shown that RRM2 is highly expressed in stem cellsin the colon (Liu 2006). At the early stages of colon cancer, RRM2expression decreases slightly. However, RRM2 expression increasessignificantly once the tumor becomes aggressive. These results supportthe findings herein that RRM2 expression is associated with an increasein cancer cell invasiveness. In addition, they support the use of theinhibitors disclosed herein to inhibit the growth or proliferation ofstem cells giving rise to cancer, thereby preventing or slowing theonset of certain cancer types.

In addition to cancer, the inhibitors disclosed herein may be used totreat other conditions associated with RR or RR overexpression, such asfor example various mitochondrial, redox-related, or degenerativediseases. In addition, the inhibitors may be used to inhibit growth orproliferation of cells expressing RR.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

EXAMPLES Example 1 Confirmation of RRM2 as a Target for Anti-CancerTherapy

An RRM2-luciferase fusion construct was co-administered with RRM2 siRNA(siRNAB+5) by hydrodynamic tail vein injection (HPTV) [plasmid 0.25mg/kg, siRNA 1.25 mg/kg] to female BALB/c mice implanted with humanHepG2 liver cancer cells. In vivo bioluminescence imaging revealedpotent down-regulation within mouse liver cancer cells over multipleweeks (FIG. 1). The tumor growth inhibition in the treatment group wassignificantly better than that observed in the control group, withplasmid alone, or with siCON. These results suggest that RRM2 playscritical role in cancer growth and validates it as a therapeutic target.

RRM2 mRNA expression levels were measured by RT-PCR in 35 human freshfrozen breast cancer biopsies and corresponding normal tissue samples.Optimal PCR primer and probe concentrations of RRM2 and β-actinhousekeeping gene were determined to reach maximum efficiency during theamplification. The PCR reaction was performed in a 20 μl final volume,adding 1 μL cDNA from each sample using Taqman PCR mix (AppliedBiosystems, Foster City, Calif.). A significant increase in RRM2expression was observed in breast cancer tissue versus correspondingnormal tissue (p<0.05) (FIG. 2A). A Western blot revealed that normalhuman tissues other than fetal liver and testis express low levels ofRRM2, whereas cancer cells express significantly higher levels of RRM2(FIG. 2B).

Human oropharyngeal cancer KB (wild-type p53) and human prostate cancerPC3 (truncated p53) cells were transfected with sense RRM2 (KBM2 andPC3M2, respectively) and control vector, and the resultingoverexpression of RRM2 was confirmed by Western blot analysis.Transfectants were applied to the upper layer of Matrigel in a Bordenchamber. After 72 hours, cells that invaded to the lower layer werefixed with alcohol, stained with methylene blue, and counted andexamined. RRM2 transfected cells exhibited increased invasive potentialin comparison to non-transfected cells (FIG. 3).

Example 2 Identification of Novel RR Inhibitors

A diverse compound library from NCI Developmental Therapeutics Program(DTP) was subjected to a virtual screening process to identify potentialRR inhibitors. The DTP library contains 2,000 different compounds. Anovel ligand binding pocket on human RRM2 identified from the X-raycrystal structure (PDB 2UW2) was selected to identify potentialinhibitor compounds that were in close proximity to the RRM1/RRM2interface but distant from the dityrosyl-diiron center in order to avoidiron chelating side effects. This ligand binding pocket, which consistsof 32 amino acid residues conserved among human and mouse RRM2 proteinfamilies, is in close proximity to the RRM1/RRM2 interface. Thestructure of the ligand binding pocket is set forth in FIG. 4. Thepocket consists of helices α7, α8, and α10 at the C-terminal domain. Thenarrow interior end of the V-shaped pocket is lined up with hydrophobicresidues near the back of dityrosyl diiron cluster center. Polarresidues such as D271, R330, and E334 that are located near the open-endof the pocket may potentially interact with the flexible C-terminus. Thepocket is lined mostly with interior hydrophobic residues with chargedresidues exposed to the surface.

Compounds that docked into the ligand binding pocket were identifiedusing the TRIPOS FlexX docking tool and ranked using an embeddedconsensus docking score. The top 80 RR inhibitor candidates thatexhibited a binding affinity equal to or greater than that of 3-AP inthe virtual screen were subjected to an in vitro screen using a knownsemi-high throughput holoenzyme-based assay for determining the potencyand subunit-selectivity of small molecule inhibitors (Shao 2005). Theassay utilized recombinant RRM1/RRM2 or RRM1/RRM2 complex and measured[³H]CDP reduction activity (i.e., CDP to dCDP) by HPLC. Ten compoundsexhibited the ability to inhibit native RRM1/RRM2 activity by greaterthan 50% in vitro, and four of these compounds inhibited enzyme activityby 80% or more. Three of the four compounds exhibiting ≧80% inhibitionshared a similar structural scaffold (NCI-3, NSC#659390, and NSC#45382),and also showed better solubility and lower toxicity than the othertested compounds. NCI-3 (dihydroxyphenylthiazole, DHPT) has thefollowing structure:

A series of NCI-3 analogs were synthesized using the strategy set forthin FIG. 5. An additional 24 NCI-3 analogs were developed by attaching avariety of R groups to the aminothiazole group. Compounds generated inthis manner included COH1, COH2, COH4, COH20 and COH29.

Example 3 Characterization of Novel NCI-3 Analogs

The ability of NCI-3 and the NCI-3 analogs synthesized in Example 2 toinhibit RR activity was tested using the in vitro holoenzyme assaydescribed above. COH4 exhibited significant RR inhibition. COH20 waseven more effective, causing 90.2% inhibition of the recombinantRRM1/RRM2 complex in vitro (FIG. 6). Results for various compounds areset forth in Table 1.

TABLE 1 Compound IC50 ± S.D. (μM) HU 148.0 ± 7.34  3-AP  1.2 ± 0.13NCI-3 19.1 ± 0.43 COH4 15.3 ± 1.8  COH20 9.3 ± 2.3

In striking contrast to 3-AP, inhibition of RR by COH20 was virtuallyunaffected by the addition of iron (Table 2).

TABLE 2 IC50 ± S.D. (μM) RRM1/RRM2 COH20 alone 9.31 ± 2.3 COH20—Fecomplex 9.12 ± 1.9 COH20—Fe complex with added Fe 10.41 ± 2.1 

Site-directed mutagenesis, Biacore analysis, and NMR Saturation TransferDifference (STD) analysis were carried out to validate the bindingpocket and ligand/protein interaction between COH20 and RRM2. RRM2 pointmutants were generated by mutating certain key residues in the bindingpocket. These residues included Y323, D271, R330, and E334, each ofwhich are charged and reside on the surface of the binding pocket, andG233, which sits deep in the pocket. Attenuation of inhibition in thesemutants confirmed involvement of the mutated residues in ligand bindingand validated the binding pocket. Interestingly, the only mutation thatdid not attenuate inhibition was G233V. This suggests the presence of ahydrophobic pocket that is stabilized by introduction of a valine sidechain.

To confirm that the ability of COH20 to inhibit RR activity was notspecific to recombinant RR, an assay was performed testing the effect ofCOH20 on intracellular RR. KB cells treated with 10 μM COH20 were lysed,and protein was extracted in a high salt buffer and passed through a G25Sephadex column to remove small molecules such as dNTP. The eluate wasmixed with [³H]CDP in reaction buffer to monitor RR activity. Treatmentwith COH20 decreased intracellular RR activity by approximately 50%(FIG. 7). Treatment with COH20 had no effect on RRM2 protein levels asmeasured by Western blot, indicating that the effect of COH20 on RRactivity is not due to a decrease in RRM2 expression.

dNTP pools from KB cells were measured by polymerase template assayfollowing treatment with 10 μM COH20. Pre- and post-treatment cellpellets were mixed with 100 μl of 15% trichloroacetic acid, incubated onice for ten minutes, and centrifuged at high speed for five minutes.Supernatants were collected and extracted with two 50 μl aliquots ofFreon/trioctylamine (55%/45%) to neutralize the trichloroacetic acid.After each addition, the samples were centrifuged at high speed andsupernatant was collected. Two 5 μl aliquots (one for each duplicate) ofeach sample were used to check dATP, dCTP, dGTP, and dTTPconcentrations. The reaction mixture in each tube contained 50 mMTris-HCL pH 7.5, 10 mM MgCl, 5 mM DTT, 0.25 mM template/primer, 1.25 μM³H-dATP (for dCTP assay) or ³H-dTTP (for dATP assay), and 0.3 units ofSequenase (2.0) in a total volume of 50 μL. DNA synthesis was allowed toproceed for 20 minutes at room temperature. After incubation, 40 μl ofeach reaction mixture was spotted onto a Whatman DE81 ion exchange paper(2.4 cm diameter). The papers were dried for 30-60 minutes at roomtemperature, washed with 5% Na₂HPO₄ (3×10 minutes), and rinsed once withdistilled water and once more with 95% ethanol. Each paper was dried anddeposited in a small vial, and 5 ml of scintillation fluid was added toeach vial. Tritium-labeled dNTPs were counted using liquid scintillationcounter and compared to standards prepared at 0.25, 0.5, 0.75, and 1.0pmol/μL of dNTPs. For comparison, duplicate sets of reactions werecarried out with freshly added inhibitors. COH20 was found to decreasedATP, dCTP, dGTP, and dTTP pools in KB cells, indicating that inhibitionof RR results in a concomitant decreased in dNTP production (FIG. 8).Similar experiments will be performed using other cell lines.

The in vitro cytotoxicity of COH4 and COH20 towards human leukemia REHand MOLT-4 cells, human prostate cancer LNCaP cells, human oropharyngealcancer KB cells, and normal fibroblast NHDF cells was evaluated using anMTT assay. 5,000 cells were seeded on six-well plates for 72 hours withvarious concentrations of drug. COH20 was cytotoxic to the cancer celllines at less than 10 μM, while causing less cytotoxicity to normalcells than 3-AP. The results are summarized in Table 3. Results forLNCaP, KB, and NHDF are set forth in FIGS. 9-11. Based on the broadrange of cancer cell types against which COH20 exhibits cytotoxicity,COH20 is expected to be cytotoxic to a variety of additional cancer celltypes, including colon cancer, breast cancer, lung cancer, melanoma,leukemia, and lymphoma cells.

TABLE 3 IC50 (μM) Cell line COH20 COH4 3-AP HU REH 2.54 20.6 1.42 32.8MOLT-4 5.26 11.85 1.21 165 LNCaP 8.49 22.96 1.75 280 KB 9.25 30.6 1.98300 NHDF 82.8 52.6 7.35 >1000

In vitro cytotoxicity assays were repeated using KBHURs, an HU-resistantclone derived from KB cells that overexpresses RRM2. COH20 was cytotoxicto KBHURs at significantly lower concentrations than the other RRinhibitors tested, confirming that COH20 is capable of overcoming HUresistance (FIG. 12). In addition, COH20 was found to be cytotoxic toKBMDR, a KB clone that overexpresses the MDR pump on the cell membrane,at lower concentrations than 3-AP or HU (FIG. 13). A real-timeproliferation assay confirmed that COH20 also inhibits cellproliferation in KBMDR cells (FIG. 14). Similar experiments will berepeated using the gemcitabine resistant cell line KBGem, which alsooverexpresses RRM2. Based on the results with other cell lines, it isexpected that COH20 will also exhibit cytotoxicity and growth inhibitiontowards KBGem.

The in vitro cytotoxicity of COH29 towards a panel of human cancer celllines was tested using the MTT assay described above. COH29significantly inhibited growth across a broad range of cancer celltypes, with an IC₅₀ of less than about 10 μM in all cell types testedexcept for colon cancer HT29, melanoma UACC-257, ovarian cancerNCI/ADR-RES, and renal cancer CAKI-1 (FIGS. 20-24). Representativeresults are summarized in Table 4.

TABLE 4 Cell line IC50 Leukemia CCRF-CEM 2.8 μM Leukemia MOLT-4 2.5 μMLeukemia SUP B15 5.0 μM Ovarian Cancer OV 90 2.6 μM

Flow cytometry and annexin staining were performed on KB cells treatedwith COH20 at 9 or 27 μM or 3-AP at 3 μM for 24 hours. These resultsshowed that COH20 treatment arrests cells in S-phase in a dose-dependentmanner (FIG. 15, upper panel). After treatment with COH20 for 72 hours,annexin staining showed significant cell death, indicating apoptosis(FIG. 15, lower panel). COH20 induced apoptosis with approximately thesame potency as 3-AP.

COH20 was injected into three male rats at 1 mg/kg for single-dosepharmacokinetic evaluation. Elimination of COH20 from plasma was foundto be tri-exponential, with a rapid initial decline phase (possibletissue distribution or liver uptake) followed by an intermediate phase(combined distribution and elimination) and a slower terminal phase(elimination) (FIG. 16). The terminal half-life (T_(1/2)) wasapproximately 5.5 hours. More detailed pharmacokinetic studies will beperformed with various dosages of COH20 to establish parameters such asclearance, bioavailability, and tissue/plasma partition coefficients.

Pharmacokinetic evaluation was performed on COH29 using the sametechniques, with COH29 administered at a dosage of 25 mg/kg. Resultsfrom triplicate analysis are summarized in Table 5. Area under the curve(AUC) calculations showed COH29 acting in a dose-dependent manner whenadministered by i.v. bolus (FIG. 25).

TABLE 5 C_(max) T_(max) AUC CL V_(ss) T_(1/2) (ng/ml) (hr) (ng*h/ml)(ml/(h*kg)) (ml/kg) (hr) 1 31300 0.03 3668 6816 1185 0.4 2 26000 0.032861 8738 576 7.6 3 25600 0.03 3918 6381 653 0.03 Avg. 27633 0.03 34827312 804 2.67 SD 1837 — 319 724 191 2.45

In order to determine the maximal tolerated dose of COH20, COH20 wasadministered to mice intravenously at dosages ranging from 10 to 160mg/kg. Other than one mouse that died in the 160 mg/kg group, bodyweight remained stable for all treatment groups, indicating that COH20is a tolerable compound with minimal toxicity (FIG. 17). There was noevidence of lethal iron chelation or induction of methemoglobulin,further indicating that COH20 has no significant iron chelation sideeffects.

A Biacore T100 instrument was used to study the ligand-proteininteraction between COH20 or 3-AP and RRM2. Wild-type RRM2 was isolatedand immobilized onto CM4 sensor chips using standard amine-couplingmethods at 25° C. and a flow rate of 10 μL/min. Specifically, thecarboxymethyl dextran surfaces of the flow cells were activated with a7-min injection of a 1:1 ratio of 0.4 M(N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.1 MN-hydroxysuccinimide (NHS). RRM2 was diluted in 10 mM sodium acetate, pH4.5, to 25 μg/ml and injected over target flow cell for targetimmobilization 8000RU. 10 mM sodium acetate, pH 4.5 buffer was injectedinto the reference flow cell for blank immobilization. The remainingactivated surface was blocked with a 7-min injection of 1Methanolamine-HCl, pH 8.5. Phosphate-buffered saline (PBS) was used as arunning buffer during immobilization. COH20 and 3-AP were dissolved inDMSO to prepare 100 mM stock solutions. The compounds were then dilutedserially in running buffer (PBS, 1.5% DMSO, 10-fold carboxymethyldextran, 0.002% Methyl-6-O-(N-heptylcarbamoyl)-α-D-glucoyranoside) tothe appropriate running concentrations. Samples were injected over thereference flow cell and target flow cell (with immobilized RRM2) at aflow rate of 60 μL/min at 25° C. Association and dissociation weremeasured for 180 seconds and 60 seconds, respectively. All compoundswere tested in triplicate at five different concentrations.Concentration series for each compound were done at least twice. Withina given compound concentration series, the samples were randomized tominimize systematic errors. Between samples, the sensor chip wasregenerated by injection of 0.3% SDS for 30 seconds. The results showeda significant interaction between COH20 and RRM2, but not between COH20and 3-AP (FIG. 18).

The Biacore T100 was also used to analyze the ability of COH20 tointerfere with binding of RRM2 to RRM1. Fixed concentrations of RRM1 (1μM) in the absence and presence of a two-fold dilution concentrationseries of COH20 (3.125-25 μM) were injected over a reference flow celland a target flow cell (with immobilized RRM2) at a flow rate 30 μL/minat 25° C. Association and dissociation were measured for 90 seconds and60 seconds, respectively. Duplicates runs were performed using the sameconditions. Between samples, the sensor chip was regenerated byinjection of 0.3% SDS, 0.2 M Na₂CO₃ for 30 seconds. COH20 was found tointerrupt the RRM1/RRM2 interaction at the interface (FIG. 19).

The cytotoxic efficacy of COH20 will be tested in vivo using a mousexenograft model. Xenograft tumor models will be created using humancancer cell lines such as KB, KBHURs, and KBGem. For establishment ofthe KB xenograft model, 1-5 10⁶ KB cells in a volume of 0.1 ml salinewill be injected into the right hind flank of 5-6 week old nude femalemice. Tumor volume will be monitored twice weekly using digitalcalipers. When tumor volume reaches approximately 100-160 mm³, mice willbe divided into groups of ten such that the median and mean body weightand tumor volume are roughly the same for all mice within a group. COH20will be administered either 1) alone in a single dose to determineeffective dosage, 2) alone at various intervals for a scheduling study,or 3) in combination with known cancer therapeutics such aschemotherapeutics. During a monitoring period of approximately fourweeks, changes in tumor cell growth, body weight, organ dysfunction, andiron chelating side effects will be analyzed at various timepoints.Following the monitoring period, mice will be euthanized and tissue,tumor, and plasma will be analyzed by visual and histologicalexamination. Based on the cytotoxicity of COH20 towards various cancercell lines in vitro, it is expected that mice treated with COH20 willexhibit higher survival rates, decreased tumor growth, and fewertumor-related side effects (e.g., weight loss, organ dysfunction).

The structure of NCI-3 analogs such as COH20 and COH29 may be refinedand optimized by generating various analogs and analyzing their bindingto RRM2 and the RRM1/RRM2 complex using site-directed mutagenesisstudies, Biacore analysis, and NMR STD experiments. X-raycrystallography studies may be performed to determine thethree-dimensional structure of the COH20-RRM2 complex. Using thesetools, additional RR inhibitors may be generated with higher potency,greater selectivity, and lower toxicity.

As shown above, the RRM2 mutant G233V enhanced COH20 inhibitor activity.Additional hydrophobic interactions between the valine side chain andthe bound ligand are believed to contribute to enhanced binding andinhibition, suggesting that an additional hydrophobic side chainextending from COH20 could optimize binding affinity. Therefore, COH20analogs that contain these hydrophobic side chains (e.g. COH1, COH2,COH4, COH29, compounds having Structure I, and compounds selected fromGroup I) are also likely to be RR inhibitors.

As stated above, the foregoing are merely intended to illustrate thevarious embodiments of the present invention. As such, the specificmodifications discussed above are not to be construed as limitations onthe scope of the invention. It will be apparent to one skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is understoodthat such equivalent embodiments are to be included herein. Allreferences cited herein are incorporated by reference as if fully setforth herein.

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1. A method of inhibiting ribonucleotide reductase activity in a cellcomprising contacting said cell with a compound selected from the groupconsisting of:

or a pharmaceutically acceptable stereoisomer or salt thereof, wherein Xis selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups; R₁-R₂are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; and R₁-R₂ may combine together to form a ring wherein thering is aryl or non-aryl.
 2. A method of inhibiting growth orproliferation of a cell expressing RRM2 comprising contacting said cellwith a compound selected from the group consisting of:

or a pharmaceutically acceptable stereoisomer or salt thereof, wherein Xis selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups; R₁-R₂are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; and R₁-R₂ may combine together to form a ring wherein thering is aryl or non-aryl.
 3. A method of treating cancer in a subject inneed thereof comprising administering to said subject a therapeuticallyeffective amount of a compound selected from the group consisting of:

or a pharmaceutically acceptable stereoisomer or salt thereof, wherein Xis selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups; R₁-R₂are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; R₁-R₂ may combine together to form a ring wherein the ringis aryl or non-aryl; and the cancer being treated is selected from thegroup consisting of lung, colon, breast, ovarian, leukemia, renal,melanoma, prostate, and CNS cancer.
 4. The method of claim 3, whereinsaid cancer is characterized by overexpression of RRM2.
 5. The method ofclaim 3, wherein said cancer is resistant to treatment with hydroxyurea.6. A method of inhibiting proliferation of a stem cell expressing RRM2in a subject in need thereof comprising administering to said subject atherapeutically effective amount of a compound selected from the groupconsisting of:

or a pharmaceutically acceptable stereoisomer or salt thereof, wherein Xis selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups; R₁-R₂are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; and R₁-R₂ may combine together to form a ring wherein thering is aryl or non-aryl.
 7. The method of any of claims 1-6, whereinsaid compound has a structure selected from the group consisting of:

or a pharmaceutically acceptable stereoisomer or salt thereof.
 8. Themethod of any of claims 1-6, wherein said compound has the structure:

or is a pharmaceutically acceptable stereoisomer or salt thereof.